Our goal is to learn the roles of histone subtypes and histone modifications in the generation of structural and functional diversity in chromatin during the cell cycle, non-dividing cell formation, and embryonic development. To unravel the complex relationship between H1 subtypes and H1 phosphorylations, we will a) locate the phosphorylation sites in each mammalian and sea urchin H1 subtype by isolating and identifying phosphopeptides, b) determine the occupancy of each phosphorylation site during different phases of the cell cycle in mammalian cells and sea urchin embryos, c) study the relation of individual phosphorylation sites to the configuration of H1 in S-phase and mitotic chromatin by means of crosslinking experiments, and d) test the hypothesis that unmodified H1 subtypes differ in their effects on chromatin conformation, by studying a variety of properties of chromatin preparations reconstituted from individual subtypes. The contribution of subtype intermixing to chromatin diversification will also be explored, by crosslinking chromatin labeled with amino acids during the embryonal switch in synthesis from one histone subtype set to another. Such studies will also provide information about the mechanism of chromatin assembly during DNA replication. Lastly, to analyze the embryonal histone switches in a single cell type, we will examine the histone subtypes synthesized in the progeny of small micromeres during their developmentally programmed transitions to the quiescent state and back to the dividing state. Our studies will provide insights into molecular events underlying developmental processes, and are pertinent to the understanding of defects in development and growth control.